Recognition by and escape from CD8+ T cell immune surveillance in cancer bearing hosts

Abstract: CD8+ T cells ( TCD8+ ) can specifically recognize tumor cells through MHC class I molecules associated with peptides that are 8-l0 amino acid residues long. In recent years, a number of tumor-associated antigens, or their epitopes, have been identified and charactenzed with the advent of new methods in molecular biology and in peptide isolation. However, cancer often grows progressively, resulting in resistance to MHC class l-restricted tumor-specific T cells. Furthermore, an impaired immune response is frequently observed in cancer-bearing hosts at an advanced stage of disease. This thesis addresses the recognition of tumor cells by autologous tumor-specific, MHC class I-restricted CD8+ T cells. and the mechanisms of escape from tumor-specific T cells. Autologous tumor-specific TCD8+ lines were generated against methylcholanthrene (MC)-induced mouse sarcomas. These tumors are known to carry unique tumor-specific antigens, and so TCD8+ against one specific tumor will not react to another. Tumor specificity was confirmed using tumor-specific TCD8+ lines in TNF-a release assays, concurring with in vivo rejection tests. Peptides eluted from MC sarcomas under mild acid conditions were fractionated by HPLC, and tested for their ability to sensitize the presentation-defective mutant RMA-S. Only one HPLC fraction from each of the three different tumor-denved peptide eluates capacitated RMA-S to induce TNF-a from specific TCD8+ lines. A different Kb-restricted peptide fraction was active for each of the three MC sarcomas, indicating that they all expressed individually distinct peptide epitopes recognized by specific TCD8+ lines. Moreover, using tumor rejection experiments in CD28-knockout mice and in vitro tumor-specific TCD8+ lines derived from CD28-knockout mice, I have shown that CD 28 co-stimulation is not required for the rejection of autologous MC tumors in hyperimmunized mice and for the recognition of autologous tumor by specific TCD8+ lines in vitro. In order to study the mechanisms by which human cancer cells escape recognition by TCD8+, nine HLA A2 restricted, autologous tumor-specific TCD8+ clones were isolated from ovarian cancer patients. Of these, two clones recognized the previously defined HER-2/neu epitope E75 (KIFGSLAFL), and one recognized the C85 epitope (ELVSEFSRM). Following co-culture of the parental tumor cells and autologous TCD8+ clones, three different escape variants were produced from an ovarian tumor line overexpressing HER-2/neu. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER-2/neu, ICAM-I or LFA-3 molecules was found. There was a correlation between the level of tumor recognition and HLA A2 expression among the tumor clones isolated from two escape variants. In contrast, high HLA A2-expressing tumor-clones isolated from one escape variant were not recognized by the HER-2/neu-specific TCD8+ clone. Collectively, these results underlines the importance of decreased expression of the HLA restriction element for escape from tumor-specific TCD8+, but also demonstrates that additional mechanisms, independent of decreased HLA class I expression, exist. Additional studies were performed with monocyte-derived DC-like cells, to develop a more sensitive assay for the detection of new peptide epitopes. Monocyte-derived DC-like cells displayed superior APC activity in their ability to elicit TNF-a release from ovarian tumor-specific TCD8+ clones. The immunosuppressive state of the tumor bearing host with advanced cancer is a well-known phenomenon. Signal transducing T cell receptor ( TCR ) ~ molecules are involved in the activation of T cells and in the assembly of the TCR-CD3 complex. I have shown that the expression of CD3 molecules is decreased in PBL from patients with cervical cancer when compared to PBL from healthy donors. Moreover, this reduced expression of ~ molecules is associated with reduced cellular functions such as the production of TNF-a. As to the mechanism involved in the reduced expression of ~ molecules, I have shown that hydrogen peroxide produced by tumor-associated macrophages or LPS activated monocytes derived from PBL can down-modulate the CD3 ~ expression, paralleling the impaired Ca2+ mobilization and reduced tumor-specific cytolytic activity of CTLs. My data suggest that macrophage-derived reactive oxygen metabolites contribute to alterations in signal transducing molecules of T cells, and to the mechanisms of immunosuppression in individuals with cancer. Key word: methylcholanthrene-induced tumor, CD8+ T cell, TCR ~ chain, HER-2/neu ISBN 91-628-2523-2